Pyrrolobenzodiazepines as antiproliferative agents

ABSTRACT

Novel pyrrolobenzodiazepines (PBDs) having a (1-methyl-1H-pyrrol-3-yl)phenyl based amino acid residue and use thereof as antiproliferative agents are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/GB2013/051098 filed Apr. 30, 2013, and claims the benefit of U.S. Provisional Application No. 61/640,310 filed Apr. 30, 2012, which is incorporated by reference herein.

The present invention relates to pyrrolobenzodiazepines (PBDs) and in particular to PBD monomers with a 4-(1-methyl-1H-pyrrol-3-yl)benzyl based amino acid residue containing substituent and methods of synthesising PBD monomers.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 35, 972-978 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:

They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents. The synthesis of the compounds has been reviewed in Thurston, D. E., et al., Chem. Rev., 1994, 94, 433-465 and Thurston, D. E., et al., Chem. Rev., 2011, 111, 2815-2864.

A number of conjugates of PBD with pyrroles and imidazoles have been reported, such as:

where n=1-3 (Damayanthi, Y., et al., Journal of Organic Chemistry, 64(1), 290-292 (1999));

where n=1-3 and

where n=1-2 (Kumar, R. and Lown, J. W. Oncology Research, 13(4), 221-233 (2003)); Kumar, R., et al., Heterocyclic Communications, 8(1), 19-26 (2002));

where n=1-4, (Baraldi, P. G., et al., Journal of Medicinal Chemistry, 42(25), 5131-5141 (1999));

where n=3, (Wells, G., et al., Proc. Am. Assoc. Canc. Res., 2003, 44, 452).

In WO 2007/039752 and Wells, G, et al., Journal of Medicinal Chemistry 2006, 49, 5442-5461, the following compound (GWL-78)

and related structures were disclosed in work by some of the present inventors. This compound showed an up to 50-fold increase in DNA binding affinity compared to its constituent PBD and dipyrrole components.

In WO 2005/085177, some of the present inventors disclosed amino acids comprising a biaryl core that could have useful properties in DNA binding.

The inventors have now discovered that the properties, particularly cytoxicity and DNA binding, of the prior art PBD conjugates can be improved. In particular, the present invention relates to the incorporation of a single 4-(1-methyl-1H-pyrrol-3-yl)benzyl based amino acid residue in combination with a single heteroaryl based amino acid residue in a PBD conjugate results in highly effective compounds.

A first aspect of the present invention provides a compound of formula I:

or a salt or solvate thereof, wherein:

the dotted double bond indicates the presence of a single or double bond between C2 and C3;

R² is selected from —H, —OH, ═O, ═CH₂, —CN, —R, OR, halo, dihalo, ═CHR, ═CHRR′, —O—SO₂—R, CO₂R and COR;

R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo;

where R and R′ are independently selected from optionally substituted C₁₋₇ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups;

R¹⁰ and R¹¹ either together form a double bond, or are selected from H and OR^(Q) respectively,

where Q is selected from O, S and NH and R^(Q) is H or C₁₋₇ alkyl or H and SO_(x)M, where x is 2 or 3, and M is a monovalent pharmaceutically acceptable cation;

A is either:

where X and Y are selected from: CH and NMe; COH and NMe; CH and S; N and NMe; N and S;

B is either a single bond or:

where X and Y are as defined above; and

R¹ is C₁₋₄ alkyl.

Thus, B1 can have the following structures:

X Y B1 CH NMe

COH NMe

CH S

N NMe

N S

A second aspect of the present invention provides a method of synthesis of a compound of formula I.

A third aspect of the present invention provides a pharmaceutical composition comprising a compound of the first aspect of the invention and a pharmaceutically acceptable carrier or diluent.

A fourth aspect of the present invention provides a compound of the first aspect of the invention for use in a method of therapy.

A fifth aspect of the present invention provides the use of a compound of the first aspect of the invention in the manufacture of a medicament for the treatment of a proliferative disease. This aspect also provides a compound of the first aspect for use in a method of treatment of a proliferative disease.

A sixth aspect of the present invention provides a method of treatment of a patient suffering from a proliferative disease, comprising administering to said patient a therapeutically acceptable amount of a compound of the first aspect or a composition of the third aspect.

In the fourth to sixth aspects of the invention, the compound of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. In the third aspect of the invention, the pharmaceutical composition may comprise one or more (e.g. two, three or four) further active agents.

Definitions

Substituents

The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substitutents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₇ alkyl: The term “C₁₋₇ alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 7 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl (C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl (amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), and neo-pentyl (C₅).

C₂₋₇ Alkenyl: The term “C₂₋₇ alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl, —CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄), pentenyl (C₅), and hexenyl (C₆).

C₂₋₇ alkynyl: The term “C₂₋₇ alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (ethinyl, —C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₇ cycloalkyl: The term “C₃₋₇ cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from:

-   -   saturated monocyclic hydrocarbon compounds:         cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),         cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),         dimethylcyclopropane (C₅), methylcyclobutane (C₅),         dimethylcyclobutane (C₆), methylcyclopentane (C₆),         dimethylcyclopentane (C₇) and methylcyclohexane (C₇);     -   unsaturated monocyclic hydrocarbon compounds:         cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),         cyclohexene (C₆), methylcydopropene (C₄), dimethylcyclopropene         (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),         methylcyclopentene (C₆), dimethylcyclopentene (C₇) and         methylcyclohexene (C₇); and     -   saturated polycyclic hydrocarbon compounds:         norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆ etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇); O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇); S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇); O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇); O₃: trioxane (C₆); N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆); N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆); N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆); N₂O₁: oxadiazine (C₆); O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and, N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene (C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), and pyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉), isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀), acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene (C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅), pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are not limited to:

C₉ (with 2 fused rings) derived from benzofuran (O₁), isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g., adenine, guanine), benzimidazole (N₂), indazole (N₂), benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂), benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁), benzothiazole (N₁S₁), benzothiadiazole (N₂S); C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁), benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄); C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂); C₁₃ (with 3 fused rings) derived from carbazole (N₁), dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂), perimidine (N₂), pyridoindole (N₂); and, C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁), phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁), thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂), phenazine (N₂).

The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkoxy group, discussed below), a C₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxy group), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxy group), preferably a C₁₋₇ alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkyl group. Examples of C₁₋₇ alkoxy groups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetal substituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocydyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in the case of a “cyclic” acetal group, R¹ and R², taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group.

Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocydyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂, —C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocycyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl), a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl), or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably a C₁₋₇ alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃ (t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃, and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents, for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇ alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case of a “cyclic” amino group, R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), or tertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³). Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃, —NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and —C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃, —C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇, alkyl group, and R² is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂, —OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O) NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently amino substituents, as defined for amino groups, and R¹ is a ureido substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂, —NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂, and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkylthio group), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of C₁₋₇ alkylthio groups include, but are not limited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyl disulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are not limited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH₃ and —S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, including, for example, a fluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl, mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉ (nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocycyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH₃ (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocycyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)₂OCH₃ (methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl; ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)₂CH₃ (mesylate) and —OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃), —S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂, —S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR'S(═O)₂OH, wherein R¹ is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocycyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR₁S(═O)R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphino groups include, but are not limited to, —PH₂, —P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀ aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonate substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and —P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphate substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and —OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramidite substituents, for example, —H, a (optionally substituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂, —OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidate substituents, for example, —H, a (optionally substituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂, —OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Nitrogen Protecting Groups

Nitrogen protecting groups are well known in the art Preferred nitrogen protecting groups are carbamate protecting groups that have the general formula:

A large number of possible carbamate nitrogen protecting groups are listed on pages 706 to 771 of Wuts, P. G. M. and Greene, T. W., Protective Groups in Organic Synthesis, 4^(th) Edition, Wiley-Interscience, 2007, which is incorporated herein by reference.

Particularly preferred protecting groups include Alloc, Troc, Teoc, BOC, Doc, Hoc, TcBOC, Fmoc, 1-Adoc and 2-Adoc.

Hydroxyl Protecting Groups

Hydroxyl protecting groups are well known in the art. A large number of suitable groups are described on pages 16 to 366 of Wuts, P. G. M. and Greene, T. W., Protective Groups in Organic Synthesis, 4^(th) Edition, Wiley-Interscience, 2007, which is incorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, benzoates, carbonates, and sulfonates.

Particularly preferred protecting groups include THP.

Proliferative Diseases

One of ordinary skill in the art is readily able to determine whether or not a candidate compound treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, bowel cancer, colon cancer, hepatoma, breast cancer, glioblastoma, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head and neck cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Cancers of particular interest include, but are not limited to, leukemias and ovarian cancers.

Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.

Cancers of particular interest include, but are not limited to, breast cancer (both ER positive and ER negative), pancreatic cancer, lung Cancer and leukaemia.

Methods of Treatment

As described above, the present invention provides the use of a compound of the first aspect of the invention in a method of therapy.

The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.

A compound may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs); surgery; and radiation therapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((2)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafamib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CBI-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER² expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a compound of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Dosage

It will be appreciated by one of skill in the art that appropriate dosages of the compound can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 100 mg, 3 times daily.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 150 mg, 2 times daily.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 200 mg, 2 times daily.

For the prevention or treatment of disease, the appropriate dosage of the compound of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of compound to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. An exemplary dosing regimen comprises a course of administering an initial loading dose of about 4 mg/kg, followed by additional doses every week, two weeks, or three weeks of a compound. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

Includes Other Forms

Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N⁺HR¹R²), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O⁻), a salt or solvate thereof, as well as conventional protected forms.

Isomers, Salts and Solvates

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

Preferably compounds of the present invention have the following stereochemistry at the C11 position:

Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C₁₋₇ alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may be cationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Compounds of formula I include compounds where a nucleophilic solvent (H₂O, R^(A)OH, R^(A)NH₂, R^(A)SH) adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R^(A)OH, where R^(A) is an ether substituent as described above):

These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.

These compounds may be isolated in solid form, for example, by lyophilisation.

General Synthetic Routes

Compounds of formula I where R¹⁰ and R¹¹ together form a double bond may be synthesised from compounds of formula 2:

R′¹⁰ is a nitrogen protecting group and R′¹¹ is O—R¹², wherein R¹² is H or a hydroxyl protecting group. Such techniques are well known in the art, and are described, for example, in Wuts, P. G. M. and Greene, T. W., Protective Groups in Organic Synthesis, 4^(th) Edition, Wiley-Interscience, 2007. If both nitrogen and hydroxyl protecting groups are present, these are preferably selected to be removable by the same conditions.

If this deprotection is carried out in a solvent of formula HQR^(Q), then R¹⁰ and R¹¹ will be H and QR^(Q) respectively. Alternatively, these groups may be introduced by adding the compound to a different solvent to that in which the deprotection is carried out.

The conversion of compounds of formula I as discussed above to those having R¹¹ as SO_(x)M may be achieved by the addition of the appropriate bisulphite salt or sulphinate salt, followed by a purification step. Further methods are described in GB 2 053 894, which is herein incorporated by reference.

Compounds of formula 2 can be made by the coupling of compounds of Formula 3 and Formula 4:

under standard amide bond formation conditions, e.g. in the presence of HOBt or DMAP and EDCl.

Compounds of formula 3 can be synthesised from compounds of formula 5:

where R′⁸ is a C₁₋₄ alkyl group, e.g. methyl. This deprotection of the carboxyl group may be carried out using standard means, e.g. treatment with base.

Compounds of formula 5 can be synthesised in general following the methods described in WO 00/12506 and WO 2007/039752, which are herein incorporated by reference. In particular, the butanoic acid side chain can be introduced at any stage in the synthesis, usually with appropriate protecting groups in place. For example, the side chain can be formed by coupling a protected or precursor form to a hydroxy group on the bezene ring using e.g. Mitsunobo coupling.

Compounds of formula 4 can be synthesised using the methods disclosed in WO 2005/085177, which are incorporated herein by reference. Reference is also made to the disclosure of WO 2007/039752.

DNA Binding

The ability of the compounds to bind to DNA, and in particular oligonucleotides, can be measured using an Ion Pair Reversed-Phase HPLC assay, as described in Rahman, K. M., et al., Journal of the American Chemical Society 2009, 131, 13756 and Narayanaswamy, M., et al., Analytical Biochemistry 2008, 374, 173. The DNA binding affinity can also be evaluated by using a calf-thymus DNA thermal denaturation assay, as described in Wells, G., et al., Journal of Medicinal Chemistry 2006, 49, 5442; Jenkins, T. C., et al., Journal of Medicinal Chemistry 1994, 37, 4529; and Gregson, S. J., et al., Journal of Medicinal Chemistry 2001, 44, 737.

Further Preferences

C2

It may be preferred in any of the embodiments that the C2 carbon is a sp² centre, so that when R² is selected from any of the following groups:

—H, —OH, —CN, —R, —OR, halo, —O—SO₂—R, —CO₂R and —COR

there is a double bond between C2 and C3.

When R² is selected from any of the following groups:

═O, ═CH₂, ═CHR, ═CHRR′

there cannot be a double bond between C2 and C3.

In further embodiments, there is no double bond between C2 and C3, and R² is H.

R²

R² is selected from —H, —OH, ═O, ═CH₂, —CN, —R, OR, halo, dihalo, ═CHR, ═CHRR′, —O—SO₂—R, CO₂R and COR.

In some embodiments, R² may be selected from —H, —OH, ═O, ═CH₂, —CN, —R, —OR, ═CHR, ═CRR′, —O—SO₂—R, —CO₂R and —COR.

In some embodiments, R² may be selected from —H, ═CH₂, —R, ═CHR, and ═CRR′.

In one embodiment, R² is H.

In one embodiment, R² is ═O.

In one embodiment, R² is ═CH₂.

In one embodiment, R² is ═CHR. Within the PBD compound, the group ═CHR may have either configuration shown below:

In one embodiment, the configuration is configuration (C1).

In one embodiment, R² is ═CRR′.

In one embodiment, R² is ═CF₂.

In one embodiment, R² is R.

In one embodiment, R² is optionally substituted C₅₋₂₀ aryl.

When R² is optionally substituted C₅₋₂₀ aryl, it may preferably be optionally substituted C₅₋₇ aryl or C₈₋₁₀ aryl. R² may further preferably be optionally substituted phenyl, optionally substituted napthyl, optionally substituted pyridyl, optionally substituted quinolinyl or isoquinolinyl. Of these groups, optionally substituted phenyl is most preferred.

When R² is optionally substituted C₅₋₂₀ aryl, it may preferably bear one to three substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.

Where R² is a C₅₋₇ aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably 1 or y to the bond to the remainder of the compound. Therefore, where the C₅₋₇ aryl group is phenyl, the substituent is preferably in the meta- or para-positions, and more preferably is in the para-position.

In one embodiment, R² is selected from:

-   -   where the asterisk indicates the point of attachment.

Where R² is a C₈₋₁₀ aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).

When R² is optionally substituted C₅₋₂₀ aryl, the substituents may be selected from: halo, hydroxyl, ether, formyl, acyl, carboxy, ester, acyloxy, amino, amido, acylamido, aminocarbonyloxy, ureido, nitro, cyano and thioether.

When R² is optionally substituted C₅₋₂₀ aryl, the substituents may be selected from the group consisting of R, OR, SR, NRR′, NO₂, halo, CO₂R, COR, CONH₂, CONHR, and CONRR′.

If a substituent on R² is halo, it is preferably F or Cl, more preferably Cl.

If a substituent on R² is ether, it may in some embodiments be an alkoxy group, for example, a C₁₋₇ alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C₅₋₇ aryloxy group (e.g. phenoxy, pyridyloxy, furanyloxy).

If a substituent on R² is C₁₋₇ alkyl, it may preferably be a C₁₋₄ alkyl group (e.g. methyl, ethyl, propyl, butyl).

If a substituent on R² is C₃₋₇ heterocyclyl, it may in some embodiments be C₆ nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C₁₋₄ alkyl groups.

If a substituent on R² is bis-oxy-C₁₋₃ alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.

Particularly preferred substituents for R² include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thienyl.

Particularly preferred substituted R² groups include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthienyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.

In one embodiment, R² is optionally substituted C₁₋₁₂ alkyl.

When R² is optionally substituted C₁₋₁₂ alkyl, it may be selected from:

(a) C₁₋₅ saturated aliphatic alkyl;

(b) C₃₋₆ saturated cycloalkyl;

(c)

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where the total number of carbon atoms in the R¹² group is no more than 5; (d)

wherein one of R^(25a) and R^(25b) is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and (e)

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃ alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.

When R² is C₁₋₅ saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.

When R² is C₃₋₆ saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.

When R² is

each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where the total number of carbon atoms in the R² group is no more than 5. In some embodiments, the total number of carbon atoms in the R² group is no more than 4 or no more than 3.

In some embodiments, one of R²¹, R²² and R²³ is H, with the other two groups being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

In other embodiments, two of R²¹, R²² and R²³ are H, with the other group being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl.

In some embodiments, R²¹ is H.

In some embodiments, R²² is H.

In some embodiments, R²³ is H.

In some embodiments, R²¹ and R²² are H.

In some embodiments, R²¹ and R²³ are H.

In some embodiments, R²² and R²³ are H.

When R² is

one of R^(25a) and R^(25b) is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.

When R² is

R²⁴, R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃ alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. In some embodiments, R²⁴ is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R²⁴ is selected from H and methyl.

In one embodiment, R² is halo or dihalo. In one embodiment, R² is —F or —F₂, which substituents are illustrated below as (C3) and (C4) respectively:

R² may preferably selected from ═CH₂, ═CH—R, where R is more preferably an optionally substituted C₁₋₄ alkyl group, and —R, where R is more preferably an optionally substituted C₅₋₂₀ aryl group. Particularly preferred groups for R² include ═CH₂, ═CH-Me, and an optionally substituted phenyl group.

R⁷

R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo;

R⁷ may preferably be selected from H, OR, SH, SR, NH₂, NHR, NRR′, and halo.

R⁷ may more preferably be selected from H and OR.

In some embodiments, R⁷ is OR, and more particularly OR^(7A), where R^(7A) is independently optionally substituted C₁₋₇ alkyl.

R^(7A) may be selected from optionally substituted saturated C₁₋₇ alkyl and optionally substituted C₂₋₄ alkenyl.

R^(7A) may preferably be selected from Me, CH₂Ph and allyl.

R¹⁰/R¹¹

R¹⁰ and R¹¹ either together form a double bond, or are selected from H and QR^(Q) respectively, where Q is selected from O, S and NH and R^(Q) is H or C₁₋₇ alkyl or H and SO_(x)M, where x is 2 or 3, and M is a monovalent pharmaceutically acceptable cation;

In some embodiments, R¹⁰ and R¹¹ form a double bond together.

In some embodiments, R¹⁰ is H and R¹¹ is OR^(Q). In these embodiments, R^(Q) may preferably be selected from H or Me.

In some embodiments, R¹⁰ is H and R¹¹ is SO_(x)M. x may preferably be 3, and M may preferably be Na⁺.

R¹

-   -   R¹ is C₁-4 alkyl.

R¹ may preferably be C₁₋₂ alkyl, and more preferably methyl.

A

A is either:

where X and Y are selected from: CH and NMe; COH and NMe; CH and S; N and NMe;

N and S.

In some embodiments, A is A1.

In some embodiments, A is A2.

In any of these embodiments, X and Y may preferably be selected from CH and NMe; CH and S; N and NMe; and N and S. X and Y may more preferably be selected from CH and NMe; and N and NMe.

In some embodiments X and Y are CH and NMe.

In some embodiments X and Y are N and NMe.

B

B is either a single bond or

where X and Y are as defined above, but independently selected.

In some embodiments, B is a single bond.

In some embodiments, B is B1.

In any of these embodiments, X and Y may preferably be selected from CH and NMe; CH and S; N and NMe; and N and S. X and Y may more preferably be selected from CH and NMe; and N and NMe.

In some embodiments X and Y are CH and NMe.

In some embodiments X and Y are N and NMe.

FIGURES

FIGS. 1A and 1B shows the results of an assay to determine the maximum tolerated dose of two compounds of the invention;

FIG. 2 shows the result of an assay to determine the in vivo activity of a compound of the invention; and

FIG. 3 shows the result of another assay to determine the in vivo activity of the same compound as in FIG. 2.

EXAMPLES

General Methods

Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 K using a Bruker Advance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). A pro-PBD numbering system is used for carbon and proton assignments for synthetic intermediates (i.e., based on the final tricyclic PBD ring system). Mass spectrometry data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectrometry data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminum plates (Merck 60, F₂₅₄), and flash chromatography utilized silica gel (Merck 60, 230-400 mesh ASTM). Parallel reactions were carried out using a Radleys™ Green House Synthesizer and parallel purifications were carried out using an IST Vacmaster™. For reactions carried out in parallel, solvents were evaporated using a Genevac VC 2000D (Genevac Technologies, UK). Purified compounds were freeze dried using a Heto-Lyolab 3000 freeze drier. Hydrogenation reactions were carried using UHP-60H hydrogen generator attached to a Parr hydrogenation apparatus. Synthetic building blocks were purchased from Maybridge Chemicals (UK), Bachem Chemicals (USA) and Sigma-Aldrich (UK). Reagents and solvents were purchased from Sigma-Aldrich (UK).

Synthesis of Key Intermediates

(a) Methyl 4-(4-(tert-butoxycarbonylamino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (5)

(i) 2-(trichloroacetyl)-1-methyl pyrrole (2)

A solution of N-methyl pyrrole (1) (113.06 g, 1.39 mol, 1.0 eq) in dry ether (350 mL) was added drop wise over a period of 1 hour and 10 minutes to a stirred solution of trichloroacetyl chloride (254 g, 1.39 mol, 1.0 eq) in dry ether (350 mL) in a 2 L, 3 necked flask. HCl gas produced in the reaction was removed by flushing with nitrogen. The reaction mixture was allowed to stir for 1.5 hours and progress of the reaction was monitored regularly by TLC and LCMS. After 1.5 hours the reaction was quenched using 1 M K₂CO₃ solution. The reaction mixture was extracted with ethyl acetate (3×) and the organic layers were combined and concentrated in vacuo. The crystalline residue was washed with n-hexane and finally dried under vacuum. Yield—281.18 g, 79.5%, NMR compared with literature

IR, (FTIR, v_(max)/cm⁻¹) 3299, 3121, 3008, 2954, 1789, 1674, 1521, 1406, 1206, 1100, 980, 881, 757; ¹H-NMR (CDCl₃, 400 MHz) δ 7.42 (1H, dd, J=4.4, 1.6 Hz), 6.97 (1H, t, J=1.6 Hz), 6.22 (1H, dd, J=4.4, 2.4 Hz) 3.97 (3H, s); ¹³C NMR (CDCl₃, 400 MHz): δ 133.6, 124.0, 122.4, 108.9, 38.5.

(ii) 1-(4-bromo-1-methyl-1H-pyrrol-2-yl)-2,2,2-trichloroethanone (3)

NBS (N-Bromosuccinimide, 2.36 g, 13.24 mmol, 1.0 equiv.) was added to a stirred solution of 2-(trichloroacetyl)-1-methylpyrrole (2) (3 g, 13.24 mmol, 1.0 equiv.) in anhydrous THF (35 mL) at −10° C. The reaction mixture was kept at −10° C. for 2 hours and then left to reach room temperature (ca. 4 hours). Excess THF was evaporated in vacuum and the solid was re-dissolved in a mixture of EtOAc/n-hexane (1:9). The resulting mixture was filtered through a plug of silica, and the filtrate was evaporated in vacuo. The resulting solid was recrystallised from n-hexane to give 3 (3.55 g, 88%). IR (FTIR, v_(max)/cm⁻¹): 3148, 2956, 1669 (C═O), 1458, 1215, 1189, 1062, 923, 842, 823, 748, 678; ¹H-NMR (CDCl₃, 400 MHz) δ 7.46 (1H, d, J=2.0 Hz), 6.95 (1H, d, J=1.6 Hz) 3.95 (3H, s); ¹³C NMR (CDCl₃, 100 MHz): δ 172.4, 132.8, 124.6, 132.2, 96.1, 38.7; EIMS m/z (+EI) calc. for C₇HsBrCl₃NO (M)⁺ 305.38. LCMS analysis found 306.86 (M+H)⁺.

(iii) Methyl 4-bromo-1-methyl-1H-pyrrole-2-carboxylate (4)

To a stirred solution of 1-(4-bromo-1-methyl-1H-pyrrol-2-yl)-2,2,2-trichloroethanone (3)(3.28 g, 10.74 mmol, 1 eq.) in dry MeOH (30 mL), a solution of sodium methoxide (0.5 mL) was added by a syringe. The sodium methoxide solution was prepared from NaH 60% in mineral oil (43 mg, 1.07 mmol, 0.1 eq.), which was previously washed with n-hexane. The solution was heated to reflux over a period of 30 minutes, when the TLC analysis showed complete consumption of the starting material. A few drops of concentrated H₂SO₄ were added to the solution to neutralise the base (pH 2). The excess MeOH was evaporated in vacuum and the resulting oil was redissolved in EtOAc (50 mL) and washed with water (40 mL). The aqueous layer was extracted with EtOAc (3×40 mL), and the organic phases were combined, dried (MgSO₄), filtered and concentrated in vacuum to afford the product as a pale white solid. (2.28 g, 97%). IR (FTIR, v_(max)/cm⁻¹): 3138, 2948, 1692, 1472, 1334, 1245, 1115, 1082, 921, 823, 753; ¹H-NMR (400 MHz, CDCl₃): δ 6.89 (d, 1H, J=2.0 Hz), 6.76 (d, 1H, J=2.0 Hz), 3.89 (s, 3H), 3.81 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃): δ 160.8, 128.7, 122.9, 119.2, 95.1, 51.2, 36.9; EIMS m/z (+EI) calc. for C₇H₈BrNO₂ (M)⁺ 218.05. found 219.26 (M+H)⁺.

(iv) Methyl 4-(4-(tert-butoxycarbonylamino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (5)

A catalytic amount of tetrakis(triphenylphosphine)palladium, Pd(PPh₃)₄ (0.477 g, 0.413, 0.06 eq) was added to a solution of 4 (1.5 g, 6.88 mmol, 1 eq) and (4-((tert-butoxycarbonyl)amino)phenyl)boronic acid (1.57 g, 6.88 mmol, 1.20 eq) in a 9:3:1 combination (13.5 ml) of EtOH, toluene and water in the presence of K₂CO₃ (2.856 g, 3 eq.) in a 10-20 mL microwave vial containing a magnetic stirrer. The reaction vessel was flushed with nitrogen during each addition. The reaction mixture was sealed in an inert N₂ atmosphere and heated with microwave radiation in an EMRYS™ Optimizer Microwave Station (Personal Chemistry) at 100° C. for 12 minutes. After LCMS and TLC analysis revealed completion of the reaction, the cooled reaction mixture was diluted with water (50 mL), extracted with EtOAc (3×40 mL), the filtrates combined, dried over MgSO₄ and concentrated under vacuum. The resulting oil was subjected to flash chromatography (n-hexane/EtOAc 9:1) to give 5 (Yield—2.2 g, 97%). IR (FTIR, v^(max)/cm⁻): 3353, 2975, 1696, 1521, 1441, 1366, 1264, 1235, 1209, 1058, 822, 799, 657; ¹H-NMR (400 MHz, CDCl₃): δ 7.40 (d, 2H, J=8.8 Hz), 7.33 (d, 2H, J=8.8 Hz), 7.16 (d, 1H, J=2.0 Hz), 7.02 (d, 1H, J=2.0), 6.45 (br s, 1H), 3.95 (s, 3H), 3.83 (s, 3H), 1.52 (s, 9H); ¹³C-NMR (100 MHz, CDCl₃): δ 161.7, 152.8, 136.5, 129.5, 125.9, 125.6, 123.7, 123.0, 119.0, 114.6, 80.5, 51.1, 36.9, 28.4; EIMS m/z (+EI) calc. for C₁₈H₂₂N₂O₄ (M)⁺ 330.38. found 330.46 (M+H)⁺.

(b) 4-(4-(tert-butoxycarbonylamino)phenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (6)

A 0.5 M solution of NaOH (2.0 eq) was added to a solution of 5 (1.0 g, 3.027 mmol) in dioxane (40 mL). The reaction mixture was allowed to stir at room temperature for 6 hours at which point TLC revealed completion of reaction. Excess 1,4-dioxane was evaporated under vacuum and the residue was diluted with water. The resulting solution was acidified with 0.5 M HCl. The product was extracted from water with 2× ethyl acetate (100 mL×2) and the organic layers were combined, washed with brine, dried over MgSO₄ and concentrated in vacuo. The product was purified using flash chromatography (ethyl acetate/n-hexane 2:8). Yield—0.92 g, 96.8%. IR (FTIR, v^(max)/cm⁻¹): 3371, 2979, 1698, 1671, 1522, 1445, 1367, 1285, 1161, 1112, 1047, 823, 803, 762, 714, 631; ¹H-NMR (400 MHz, CDCl₃): δ 8.33 (1H, s), 7.55 (d, 2H, J=8.8 Hz), 7.50 (d, 2H, J=8.8 Hz), 7.36 (d, 1H, J=2.0 Hz), 7.22 (d, 1H, J=2.0), 3.97 (s, 3H), 1.50 (s, 9H); ¹³C-NMR (100 MHz, CDCl₃): δ 162.3, 153.7, 138.6, 123.0, 127.1, 126.0, 124.4, 124.0, 119.5, 115.1, 79.9, 36.9, 28.6; EIMS m/z (+EI) calc. for C₁₇H₂₀N₂O₄ (M)⁺ 316.35. found 315.16 (M+H)⁺.

(c)

(i) Methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (7)

5 (1 g, 3.027 mmol) was dissolved in a small volume of MeOH and 4M HCl in dioxane (15 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 6 hours at which point TLC showed completion of reaction. Excess solvent was evaporated under vacuum to obtain a brown coloured solid 7. The solid product was subjected to flash chromatography (n-hexane/EtOAc 9:1) to give pure 7 (065 g, 94.2%). IR (FTIR, v^(max)/cm⁻¹): 3366, 2987, 1688, 1629, 1566, 1422, 1372, 1262, 1181, 1103, 1067, 951, 821, 784, 756; ¹H-NMR (400 MHz, CDCl₃): δ 7.28 (2H, d, J=8.4 Hz), 7.11 (1H, d, J=2.0 Hz), 6.96 (1H, d, J=2.0 Hz), 6.68 (d, 2H, J=8.0 Hz), 3.94 (s, 3H), 3.83 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃): δ 161.7, 144.7, 126.2, 125.4, 125.2, 115.5, 114.4, 51.0, 36.8; EIMS m/z (+EI) calc. for C₁₃H₁₄N₂O₂ (M)⁺ 230.26. found 231.1 (M+H)⁺.

(ii) Methyl 4-(4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (8)

0.2 g boc protected 6 (0.63 mmol, 1.2 eq) was dissolved in DMF (5 mL) to which 2.0 eq of EDCl and 2.5 eq of DMAP were added and the mixture was allowed to stir for 30 minutes. At this point methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate (80.9 mg, 0.52 mmol, 1.0 eq) was added and the reaction mixture was allowed to stir for a further 3 hours at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×50 mL). The combined extracts were sequentially washed with saturated aqueous NaHCO₃ (50 mL), water (50 mL), brine (50 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product was used for the boc deprotection step to afford 8 without further purification. For boc deprotection, the crude product was dissolved in a small volume of MeOH and 4M HCl in dioxane (5 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 2 hours at which point TLC showed completion of reaction. Excess solvent was evaporated under vacuum to obtain a brown coloured solid (8). The solid product was subjected to flash chromatography (n-hexane/EtOAc 9:1) to give pure 8. Yield 0.21 gm, 77%.

¹H-NMR (400 MHz, CDCl₃): δ 7.72 (1H, s), 7.69 (1H, s), 7.57 (2H, d, J=8.0 Hz), 7.46 (4H, d, J=8.0 Hz), 7.41 (2H, d, J=8.0 Hz), 7.20 (1H, d, J=2.0 Hz), 7.06 (1H, d, J=2.0 Hz, 7.02 (1H, d, J=1.6 Hz), 6.92 (1H, s). m/z (+EI) calc. for C₂₅H₂₄N₄O₃ (M)⁺ 428.48. found 429.26 ([M+H]⁺.

(d)

(i) Methyl 4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate (9)

Two eq of EDCl and 2.5 eq of DMAP were added to a stirred solution of 6 (0.45 gm, 1.2 eq) in DMF (8 mL) and the mixture was allowed to stir for 30 minutes after which methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate (0.18 g, 1.18 mmol, 1.0 eq) was added. The reaction mixture was allowed to stir for a further 6 hours at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×150 mL). The combined extracts were sequentially washed with citric acid (100 mL), saturated aqueous NaHCO₃ (100 mL), water (100 mL), brine (100 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product 9a (0.58 gm, yield 90.6%) was used for the boc deprotection step to afford 9 without further purification. For boc deprotection, 0.29 gm of 9a was dissolved in a small volume of MeOH and 4M HCl in dioxane (15 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 6 hours at which point TLC showed completion of reaction. Excess solvent was evaporated under vacuum to obtain a brown coloured solid (9). The solid product was subjected to flash chromatography (n-hexane/EtOAc 9:1) to give pure 9. Yield 0.21 gm, 95%.

(ii) Methyl 4-(4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate (10)

Lithium hydroxide (68 mg, 1.65 mmol, 3 eq) was added to 9a (0.25 g, 0.55 mmol) in aqueous dioxane (8 ml dioxane, 4 ml water) at room temperature. The reaction mixture was stirred for 3 hours at which point TLC showed completion of reaction. Dioxane was evaporated under high vacuum and the residue was diluted with water. The resulting solution was acidified with 1 M citric acid followed by extraction with ethyl acetate (2×50 mL). The organic layer combined and washed with brine (50 mL), dried over MgSO₄ and finally concentrated using a rotary evaporator under reduced pressure to obtain the hydrolysed acid form of 9a as a white solid (yield 0.23 g, 91.6%). To a stirring solution of the white solid (0.23 gm, 0.52 nmol) in DMF, 2.0 equivalent of EDCl and 2.5 Equivalent of DMAP was added. After stirring the mixture for 20 minutes, commercially available methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate (80.1 mg, 0.52 mmol, 1.0 eq) was added. The reaction mixture was allowed to stir for a further 3 hours at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×50 mL). The combined extracts were sequentially washed with saturated aqueous NaHCO₃ (50 mL) and brine (50 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product was used for the boc deprotection step to afford 10. For boc deprotection, the crude intermediate was dissolved in a small volume of MeOH and 4M HCl in dioxane (5 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 3 hours at which point TLC showed completion of reaction. Excess solvent was evaporated under vacuum to obtain a brown coloured solid (10). The solid product was subjected to flash chromatography (n-hexane/EtOAc 8:2) to give pure 10. Yield 0.20 gm, 83% over 2 steps. ¹H-NMR (DMSO, 400 MHz): δ 9.72 (1H, s), 8.09 (1H, t, J=5.6 Hz), 7.71 (2H, d, J=8.8 Hz), 7.49 (2H, d, J=8.8 Hz), 7.40 (1H, d, J=2.0 Hz), 7.27 (1H, d, J=2.0), 7.19 (1H, d, J=2.0), 7.03 (1H, dd, J=4.0, 1.6 Hz), 7.00 (1H, t, J=2.0 Hz), 6.84 (1H, d, J=2.0 Hz), 6.10 (1H, m), 3.89 (3H, s). m/z (+EI) calc. for C₂₅H₂N₆O₄ (M)⁺ 474.51. found 475.35 ([M+H]⁺.

(e)

Methyl 4-(4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylate (11)

0.3 gm of boc protected 6 (0.94 mmol, 1.2 eq) was dissolved in DMF (5 mL) to which 2.0 eq of EDCl and 2.5 eq of DMAP were added. The mixture was allowed to stir for 30 minutes after which methyl 4-amino-1-methyl-1H-imidazole-2-carboxylate (0.121 g, 0.79 mmol, 1.0 eq) was added. The reaction mixture was allowed to stir for a further 6 hours at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×150 mL). The combined extracts were sequentially washed saturated aqueous NaHCO₃ (50 mL), water (50 mL), brine (50 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product 11a (0.48 gm) was used for the boc deprotection step to afford 11. For boc deprotection, the crude intermediate was dissolved in a small volume of MeOH and 4M HCl in dioxane (5 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 2 hours at which point TLC showed completion of reaction. Excess solvent was evaporated under vacuum to obtain a brown coloured solid (11). The solid product was subjected to flash chromatography (n-hexane/EtOAc 9:1) to give pure 11. Yield 0.35 gm, 81% over two steps.

¹H-NMR (DMSO, 400 MHz): 9.75 (1H, s), 8.03 (1H, s), 7.71 (2H, d, J=8.8 Hz, 7.53 (1H, s), 7.52 (1H, s), 7.48 (2H, d, J=8.8 Hz), 7.42 (1H, s), 7.19 (1H, d, J=2.0), 3.94 (3H, s), 3.91 (3H, s), 3.89 (3H, s). m/z (+EI) calc. for C₁₈H₁₉N₅O₃ (M)⁺ 353.38. found 354.42 ([M+H]⁺.

(f) 4-(10-(allyloxycarbonyl)-7-methoxy-5-oxo-11-(tetrahydro-2H-pyran-2-yloxy)-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanoic acid (13)

A 0.5 M solution of NaOH (made from 1.4135 g of NaOH) was added to a solution of 12 (Compound 18, WO 2007/039752) in dioxane at room temperature. The reaction mixture was allowed to stir for 4 hours at which point TLC showed completion of the reaction. Dioxane was evaporated under high vacuum and the residue was diluted with water. The resulting solution was acidified with 1 M citric acid followed by extraction with ethyl acetate (2×100 mL). The combined organic layers were washed with brine (100 mL), dried over MgSO₄ and finally concentrated using a rotary evaporator under reduced pressure. Yield—8.7 g, (94%), ¹H-NMR (400 MHz, CDCl₃): δ 7.2 (2H, s), 6.90 (1H, s), 6.58 (1H, s), 5.85 (2H, d, J=9.2 Hz), 5.73 (2H, d, J=9.2 Hz), 5.03-5.13 (m, 6H), 4.68-4.35 (m, 4H), 4.09-4.01 (m, 4H), 3.91-3.82 (m, 8H), 3.69-3.46 (m, 8H), 2.60-2.55 (m, 4H), 2.18-2.00 (m, 10H), 1.76-1.55 (m, 4H), 1.53-1.43 (m, 8H); ¹³C-NMR (100 MHz, CDCl3): δ 177.6, 167.6, 149.8, 132.1, 131.9, 126.7, 117.3, 114.9, 110.8, 100.7, 96.0, 91.7, 88.5, 67.9, 66.6, 63.6, 60.1, 56.1, 46.5, 31.1, 30.3, 28.8, 25.2, 24.1, 23.2, 20.0; EIMS m/z (+EI) calc. for C26H34N2O9 (M)⁺ 518.56. found 519.26 (M+H)⁺.

(g) Methyl 4-[[4-[[4-(4-aminophenyl)-1-methyl-pyrrole-2-carbonyl]amino]-1-methyl-imidazole-2-carbonyl]amino]-1-methyl-pyrrole-2-carboxylate (19)

Lithium hydroxide (40 mg, 1.65 mmol, 3 eq) was added to 11a (0.25 g, 0.55 mmol) in aqueous dioxane (8 ml dioxane, 4 ml water) at room temperature. The reaction mixture was stirred for 3 hours at which point TLC showed completion of reaction. Dioxane was evaporated under high vacuum and the residue was diluted with water. The resulting solution was acidified with 1 M citric acid followed by extraction with ethyl acetate (2×50 mL). The organic layer combined and washed with brine (50 mL), dried over MgSO₄ and finally concentrated using a rotary evaporator under reduced pressure to obtain the hydrolysed acid form of 17 as a white solid (yield 0.235 g, 97%) which was used for the next reaction without any further purification. To a stirring solution 17 (0.235 gm, 0.54 nmol) in DMF, 2.0 equivalent of EDCl and 2.5 Equivalent of DMAP was added. After stirring the mixture for 20 minutes, commercially available methyl 4-amino-1-methyl-1H-imidazole-2-carboxylate (100.00 mg, 0.54 mmol, 1.2 eq) was added. The reaction mixture was allowed to stir for a further 3 hours at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×50 mL). The combined extracts were sequentially washed with saturated aqueous NaHCO₃ (50 mL) and brine (50 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product was used for the boc deprotection step to afford 18 which was dissolved in a small volume of MeOH and 4M HCl in dioxane (5 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 3 hours at which point TLC showed completion of reaction. Excess solvent was evaporated under vacuum to obtain a brown coloured solid (19). The solid product was subjected to flash chromatography (n-hexane/EtOAc 8:2) to give pure 19. Yield 0.22 gm, 85% over 2 steps.

¹H-NMR (DMSO, 400 MHz): 10.09 (1H, s), δ 9.89 (1H, s), 7.78 (2H, d, J=8.8), 7.68 (1H, s), 7.49 (2H, d, J=8.64), 7.35 (1H, d, J=1.6), 7.21 (1H, d, J=2.0 Hz), 7.15 (1H, d, J=2.0 Hz), 3.97 (6H, s), 3.90 (3H, s), 3.84 (3H, s) m/z (+EI) calc. for C₂₃H₂₃N₇O₄ (M)⁺ 461.47. found 462.17 ([M+H]⁺.

Example 1

A Q² 15/16a*  5 MPB

15/16b*  8 MPB-MPB

15/16c  9 MPB-Py

15/16d 11 MPB-Im

15/16g 10 MPB-Py-Py

15/16h 19 MPB-Py-Im

*comparative examples

(a) (S)-methyl 4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (16a) (i) (11aS)-allyl 7-methoxy-8-(4-(4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenylamino)-4-oxobutoxy)-5-oxo-11-(tetrahydro-2H-pyran-2-yloxy)-2,3,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (15a)

A solution of Alloc-THP protected PBD acid 13 (3.72 g, 7.16 mmol, 1.2 equivalent) was dissolved in DMF. EDCl (2.49 g, 13.02 mmol, 2.0 eq) and DMAP (1.989 g, 16.28 mmol, 2.5 eq) were added to the stirred solution of 13 at room temperature and the mixture was allowed to stir for 30 minutes after which the MPB-ester 7 (1.5 g, 6.514 mmol, 1.0 eq) was added. The reaction mixture was allowed to stir for a further 2 hours at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×150 mL). The combined extracts were sequentially washed with citric acid (200 mL), saturated aqueous NaHCO₃ (250 mL), water (250 mL), brine (250 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product was purified by silica gel flash chromatography (MeOH:CHCl₃, 20:80) to give a white foamy solid, 15a. Yield—4.05 g, 85.5%. (FTIR, v_(max)/cm⁻¹): 2949, 2362, 1704, 1600, 1514, 1436, 1372, 1269, 1203, 1107, 1021, 964, 765. (¹H NMR, 400 MHz, CDCl₃): δ 7.82 (1H, s), 7.48 (2H, m), 7.41 (1H, d, J=2.0 Hz), 7.40 (1H, d, J=2.4 Hz), 7.23 (2H, d, J=8.4 Hz), 7.17 (1H, d, J=2.0 Hz), 7.04 (1H, d, J=2.0 Hz), 5.93-5.65 (2H, m), 5.09-5.4.97 (m, 4H), 4.68-4.32 (m, 4H), 4.15-4.10 (m, 4H), 3.94-3.82 (m, 12H), 3.68 (m, 2H), 3.59-3.49 (m, 6H), 2.60-2.57 (m, 3H), 2.15-2.00 (m, 8H), 1.88-1.80 (m, 2H), 1.79-1.70 (6H), 1.60-1.44 (m, 12H); (¹³C NMR, 100 MHz, CDCl₃): δ 177.1, 170.5, 167.3, 161.6, 149.1, 136.3, 132.1, 131.9, 130.4, 128.9, 127.1, 125.9, 125.4, 123.5, 123.1, 120.3, 117.3, 114.6, 110.8, 91.5, 88.6, 68.2, 66.5, 64.3, 63.6, 60.3, 56.0, 51.1, 46.4, 36.8, 31.1, 30.9, 29.1, 25.1, 24.6, 23.2, 21.0, 20.1; m/z (+EI) calc. for C₃₉H₄₆N₄O₁₀ (M)⁺ 730.80. found 731.67 ([M+H]⁺.

(ii) (S)-methyl 4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (16a)

Palladium tetrakis[triphenylphosphine](5.60 mg, 4.8 μM, 0.05 equiv) was added to a solution of Alloc-THP-PBD conjugate 15a (70 mg, 0.097 mmol), pyrrolidine (8.36 mg, 0.117 mmol, 1.2 eq) and triphenylphosphine (8.62 mg, 0.25 equiv) in DCM (5 mL). The reaction mixture was stirred at room temperature for 2 hours at which point TLC showed completion of reaction. Excess DCM was removed by rotary evaporation under reduced pressure and the resulting residue dried in vacuo to remove pyrrolidone. The product was purified by column chromatography (eluted with n-hexane 65%%, EtOAc 35%) to give the product as a yellowish solid, 3.37 (40 mg, 77%). [α]^(22.7) _(D)+165° (c=0.046, CHCl3); IR (FTIR, v^(max)/cm⁻¹): 3297, 2944, 2358, 1701, 1598, 1567, 1508, 1442, 1374, 1264, 1212, 1181, 1106, 1072, 824, 730; ¹H-NMR (500 MHz, CDCl₃): δ 7.68 (1H, s), 7.65 (1H, d, J=4.5 Hz, H-11), 7.52 (1H, s, H-6), 7.46 (2H, dd, J=8.4, 2.0 Hz, 2Ar—H), 7.40 (2H, dd, J=8.4, 2.0 Hz, 2Ar—H), 7.16 (1H, d, J=2.0 Hz, Py-H), 7.03 (1H, d, J=1.6 Hz, Py-H), 6.82 (1H, s, H-9), 4.12-4.20 (2H, m, CH₂ side chain linker), 3.94 (3H, s, N—CH₃), 3.88 (3H, s, O—CH₃), 3.68-3.71 (1H, m, H-11a), 3.50-3.60 (2H, m, H2-3), 2.58-2.62 (2H, m, CH₂), 2.26-2.31 (4H, m, CH₂), 1.50-1.54 (2H, m, CH₂); ¹³C-NMR (125 MHz, CDCl₃): δ 164.5, 162.4, 161.6, 150.5, 147.8, 140.7, 125.9, 125.5 (2C), 123.6, 123.1, 120.3, 114.6, 111.8, 111.0, 94.4 (2C), 68.0, 63.7, 56.1, 53.7, 51.0, 46.6, 36.8, 31.9, 29.6, 25.2, 24.8, 24.1, 20.2; HRMS m/z (+EI) calc. for C₃₀H₃₂N₄O (M+H)⁺ 545.2400. found 545.2422 (M+H)⁺, δ 4 ppm.

Compounds 15b-d, g, h and 16b-d, g, h were made in an analogous manner, reacting compound A with 13, followed by deprotection.

(b) (S)-methyl 4-(4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (16b)

[α]^(22.7) _(D)+134° (c=0.038, CHCl₃); IR (FTIR, v_(max)/cm⁻¹): 3850.89, 3732, 3619, 2443, 2354, 2228, 2169, 2091, 1971, 1859, 1729, 1679, 1521, 1265, 734, 629; ¹H-NMR (500 MHz, CDCl₃): δ 7.72 (1H, s, NH), 7.69 (1H, s, NH), 7.66 (1H, d, J=4.0 Hz, H-11), 7.57 (2H, d, J=8.0 Hz, 2Ar—H), 7.53 (1H, s, H-6), 7.46 (4H, d, J=8.0 Hz, 4Ar—H), 7.41 (2H, d, J=8.0 Hz, 2Ar—H), 7.20 (1H, d, J=2.0 Hz, Py-H), 7.06 (1H, d, J=2.0 Hz, Py-H), 7.02 (1H, d, J=1.6 Hz, Py-H), 6.92 (1H, s, Py-H), 6.84 (1H, s, H-9), 4.12-4.20 (2H, m, CH₂ side chain linker), 4.00 (3H, s, N—CH₃), 3.96 (3H, s, N—CH₃), 3.88 (3H, s, O—CH₃), 3.84 (3H, s, O—CH₃), 3.70-3.73 (1H, m, H-11a), 3.55-3.61 (2H, m, H₂-3), 2.58-2.62 (2H, m, CH₂), 2.29-2.31 (2H, m, CH₂), 1.93-2.06 (4H, m, CH₂); ¹³C-NMR (125 MHz, CDCl₃): δ 164.5, 162.4, 161.7, 150.7, 147.3, 139.2, 126.0, 125.6, 125.4 (2C), 125.2 (2C), 123.0, 120.4 (2C), 114.6 (2C), 111.4, 94.6 (2C), 68.3, 63.7, 56.1, 51.6 (2C), 41.0, 36.9, 31.9, 29.6, 25.2, 24.2, 24.1, 20.2; HRMS m/z (+EI) calc. for C₄₂H₄₂N₆O₇ (M+H)⁺ 743.3193. found 743.3193 ([M+H]⁺, δ 0.3 ppm).

(c) (S)-methyl 4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate (16c)

[α]^(22.7) _(D)+128° (c=0.037, CHCl₃); IR (FTIR, v_(max)/cm⁻¹): 3321, 2237, 2107, 2041, 1967, 1860, 1685, 1517, 1435, 1254, 1180, 1118, 749, 722, 696, 667; ¹H-NMR (500 MHz, CDCl₃): δ 7.98 (1H, s, NH), 7.88 (1H, s, NH), 7.68 (1H, s, H-6), 7.65 (1H, d, J=4.0 Hz, H-11), 7.64 (2H, d, J=8.0 Hz, 2Ar—H), 7.54 (1H, d, J=1.6 Hz, Py-H), 7.52 (1H, d, J=1.6 Hz, Py-H), 7.45 (1H, d, J=2.0 Hz, Py-H), 7.33 (2H, d, J=8.0 Hz, 2Ar—H), 6.97 (1H, s, Py-H), 6.89 (1H, s, H-9), 4.08-4.18 (2H, m, CH₂), 3.97 (3H, s, N—CH₃), 3.89 (3H, s, N—CH₃), 3.84 (3H, s, O—CH₃), 3.79 (3H, s, O—CH₃), 3.66-3.70 (1H, m, H-11a), 3.55-3.60 (2H, m, H₂-3), 2.56-2.61 (2H, m, CH₂), 2.23-2.32 (4H, m, CH₂), 2.00-2.05 (2H, m); ¹³C-NMR (125 MHz, CDCl₃): δ 162.5, 161.6, 159.1, 150.4, 147.7, 138.4, 132.8, 132.1 131.9 (2C), 128.6, 128.4 (2C), 125.4 (2C), 124.8, 123.0, 121.0, 120.4 (2C), 116.2, 114.6 (2C), 109.9, 94.2, 67.4, 63.6, 57.1, 53.7, 51.1, 46.7, 36.9, 36.7, 34.0, 29.6, 24.2; HRMS m/z (+EI) calc. for C₃₆H₃₈N₆O₇ (M)⁺ 667.2880. found 667.2881 ([M+H]⁺, δ 0.1 ppm).

(d) (S)-ethyl 4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylate (16d)

[α]^(22.7) _(D)+122° (c=0.028, CHCl₃), IR (FTIR, v_(max)/cm⁻¹): 3324, 2355, 2157, 2109, 2032, 1913, 1600, 1533, 1465, 1262, 1179, 1109, 751; ¹H-NMR (500 MHz, CDCl₃): δ 8.47 (1H, s, NH), 7.72 (1H, s, NH), 7.66 (1H, d, J=4.0 Hz, H-11), 7.55 (1H, s, H-6), 7.52 (1H, d, J=2.0, Py-H), 7.49 (2H, d, J=8.0 Hz, 2Ar—H), 7.37 (2H, d, J=8.0 Hz, 2Ar—H), 7.16 (1H, d, J=1.6 Hz, Py-H), 7.03 (1H, s, Im-H), 6.91 (1H, s, H-9), 4.39-4.43 (2H, m, O—CH₂) 4.13-4.22 (2H, m, CH₂), 4.01 (3H, s, N—CH₃), 3.99 (3H, s, N—CH₃), 3.83 (3H, s, O—CH₃), 3.68-3.72 (1H, m, H-11a), 3.55-3.60 (2H, m, H₂-3), 2.58-2.63 (2H, m, CH₂), 2.24-2.32 (4H, m, CH₂), 2.00-2.07 (2H, m, H₂-1), 141-1.45 (3H, m, CH₃); ¹³C-NMR (125 MHz, CDCl₃): δ 163.1, 162.5, 158.7, 150.4, 147.7, 140.7, 137.2, 131.5, 125.6 (2C), 125.4 (2C), 123.8, 123.0, 120.4 (2C), 114.5, 111.6, 110.8, 109.9, 100.0, 67.4, 61.5, 56.1, 53.7, 51.1, 46.7, 37.0, 36.0, 34.0, 29.6, 24.8, 24.2, 14.4; HRMS m/z (+EI) calc. for C₃₆H₃₉N₇O₇ (M)⁺ 682.2989 found 682.2986 ([M+H]⁺, δ−0.4 ppm).

(e) (S)-methyl 4-(4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate (16g)

[α]^(22.7) _(D)+149° (c=0.054, CHCl₃); IR (FTIR, v_(max)/cm⁻¹): 3310, 2947, 2358, 2168, 2153, 2132, 2070, 2011, 1989, 1651, 1538, 1434, 1402, 1257, 1107, 753; ¹H-NMR (400 MHz, CDCl₃): δ 8.02 (1H, s, NH), 7.88 (1H, d, J=5.2 Hz, H-11), 7.68 (1H, s, H-6), 7.67 (1H, d, J=1.6 Hz, Py-H), 7.64 (1H, d, J=1.6 Hz, Py-H), 7.53 (2H, d, J=8.0 Hz, 2Ar—H), 7.45 (1H, d, J=1.6 Hz, Py-H), 7.31 (2H, d, J=8.0 Hz, 2Ar—H), 7.20 (1H, s, Py-H), 6.96 (1H, s, Py-H), 6.89 (1H, bs, NH), 6.81 (1H, s, H-9), 6.78 (1H, d, J=1.6 Hz, Py-H), 6.71 (1H, bs, NH), 4.11-4.16 (2H, m, CH₂), 3.97 (3H, s, N—CH₃), 3.92 (3H, s, N—CH₃), 3.88 (3H, s, N—CH₃), 3.84 (3H, s, N—CH₃), 3.79 (3H, s, O—CH₃), 3.68-3.71 (1H, m, H-11a), 3.55-3.60 (2H, m, H₂-3), 2.56-2.61 (2H, m, CH₂), 2.22-2.28 (4H, m, CH₂), 1.99-2.04 (2H, m); HRMS m/z (+EI) calc. for C₄₁H₄₃N₉O₈ (M)⁺ 790.3313 found 790.3314 [M+H]⁺, δ 0.1 ppm.

(f) (S)-methyl 4-(4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate (16h)

[α]^(22.7) _(D)+142° (c=0.043, CHCl₃); IR (v_(max)/cm⁻¹): 3408, 2358, 2168, 2148, 2019, 1978, 1938, 1718, 1534, 1260, 1118, 757; ¹H-NMR (500 MHz, CDCl₃): δ 8.72 (1H, s, NH), 8.12 (1H, s, NH), 7.71 (1H, s), 7.65 (1H, d, J=4.4 Hz), 7.53 (1H, s), 7.48 (2H, d, J=8.0 Hz), 7.47 (1H, s), 7.42 (2H, d, J=1.6 Hz), 7.40 (2H, d, J=8.0 Hz), 7.03 (1H, d, J=1.6 Hz), 6.95 (1H, s), 6.82 (1H, s), 6.81 (1H, d, J=1.6 Hz), 4.12-4.21 (2H, m), 4.07 (3H, s), 4.00 (3H, s), 3.91 (3H, s), 3.89 (3H, s), 3.81 (3H, s), 3.69-3.72 (1H, m), 3.55-3.61 (2H, m), 2.58-2.63 (2H, m), 2.26-2.32 (4H, m, CH₂), 2.02-2.07 (2H, m); HRMS (EI, m/z): Calc. for C₄₁H₄₃N₉O₈ (MH⁺): 790.3313. Found, 790.3314.

Example 2

Q¹ 14a

14b

Q² 15/16e Py-MPB

15/16f Im-MPB

(a)

(i) 4-(4-(((11S,11aS)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxylic acid (14a)

A solution of Alloc-THP protected PBD acid 13 (1.85 g, 3.57 mmol, 1.2 equivalent) was dissolved in DMF. EDCl (1.24 g, 6.48 mmol, 2.0 eq) and DMAP (0.99 g, 8.1 mmol, 2.5 eq) were added to the stirred solution of 13 at room temperature and the mixture was allowed to stir for 30 minutes after which methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate (0.5 g, 3.243 mmol, 1.0 eq) was added. The reaction mixture was allowed to stir for a further 6 hours at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×150 mL). The combined extracts were sequentially washed with citric acid (200 mL), saturated aqueous NaHCO₃ (250 mL), water (250 mL), brine (250 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product (1.88 gm) was used for hydrolysis reaction to afford 14a. For hydrolysis, Lithium hydroxide (0.24 g, 5.71 mmol, 3 eq) was added to the crude product (1.88 g, 2.87 mmol) in aqueous dioxane (75 ml dioxane, 11.5 ml water) at room temperature. The reaction mixture was stirred for 3 hours at which point TLC showed completion of reaction. Dioxane was evaporated under high vacuum and the residue was diluted with water. The resulting solution was acidified with 1 M citric acid followed by extraction with ethyl acetate (2×100 mL). The organic layer combined and washed with brine (100 mL), dried over MgSO₄ and finally concentrated using a rotary evaporator under reduced pressure to obtain 14a as a white solid (yield, 1.68 gm, 74.0% over 2 steps). ¹H-NMR δ 9.09 (1H, s, NH), 7.39 (1H, d, J=2.0 Hz), 7.14 (1H, s, H-6), 7.12 (1H, s, H-6), 6.96 (1H, s, H-9), 6.76 (1H, d, J=2.0 Hz, Py-H), 5.86-5.75 (2H, m, H-11), 5.13-4.84 (3H, m), 4.61-4.21 (2H, m), 4.06-3.88 (3H, m, side-chain H-1, pyran H-6), 3.87 (3H, s, O/NCH3), 3.87 (3H, s, O/NCH3), 3.86 (3H, s), 3.53-3.44 (3H, m), 2.55-2.45 (2H, m), 2.13-1.88 (6H, m), 1.70-1.39 (6H). m/z (+EI) calc. for C₃₂H₄₀N₄O₁₀ (M)⁺ 640.68. found 641.57 ([M+H]⁺.

(ii) (11S,11aS)-allyl 7-methoxy-8-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (15e)

A solution of Alloc-THP protected PBD-Py acid 14a (150 mg, 0.23 mmol, 1.0 equivalent) was dissolved in DMF. EDCl (2.49 g, 13.02 mmol, 2.0 eq) and DMAP (1.989 g, 16.28 mmol, 2.5 eq) were added to the stirred solution of 13 at room temperature and the mixture was allowed to stir for 30 minutes after which the MPB-ester 7 (67.83 mg, 0.29 mmol, 1.25 eq) was added. The reaction mixture was allowed to stir for a further 3 hour at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3×150 mL). The combined extracts were sequentially washed with citric acid (50 mL), saturated aqueous NaHCO₃ (50 mL), water (50 mL), brine (50 mL) and finally dried over MgSO₄. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product was directly used in the next step without further purification. m/z (+EI) calc. for C₄₅H₅₂N₆O₁₁ (M)⁺ 852.93. found 854.87 ([M+H]⁺.

(iii) (S)-methyl 4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (16e)

Palladium tetrakis[triphenylphosphine](12.17 mg, 10.5 μM, 0.05 equiv) was added to a solution of Alloc-THP-PBD conjugate 15e (179 mg, 0.21 mmol), pyrrolidine (17.91 mg, 0.25 mmol, 1.2 eq) and triphenylphosphine (13.81 mg, 0.25 equiv) in DCM (5 mL). The reaction mixture was stirred at room temperature for 2 h at which point TLC showed completion of reaction. Excess DCM was removed by rotary evaporation under reduced pressure and the resulting residue dried in vacuo to remove pyrrolidone. The product was purified by high performance liquid chromatography (eluted with acetone:water gradient with 1% TFA) to give the product as a light yellowish solid, 16e (48 mg, 34% after HPLC purification).

[α]^(22.7) _(D)+197° (c=0.052, CHCl₃), IR (FTIR, v_(max)/cm⁻¹): 3330, 2360, 2214, 2180, 2041, 2020, 1999, 1967, 1698, 1517, 1438, 1265, 1180, 1119, 756, 722, 696, 667, 630; ¹H-NMR (500 MHz, CDCl₃): δ 7.77 (1H, s, NH), 7.68 (1H, s, H-6), 7.67 (2H, d, J=8.0 Hz, 2Ar—H), 7.64 (1H, d, J=5.0 Hz, H-11), 7.55 (2H, d, J=8.0 Hz, 2Ar—H), 7.47 (1H, d, J=2.0 Hz, Py-H), 7.43 (1H, s, NH), 7.18 (1H, d, J=2.0 Hz, Py-H), 7.09 (1H, d, J=2.0 Hz, Py-H), 7.05, (1H, d, J=2.0 Hz, Py-H), 6.83 (1H, s, H-9), 4.09-4.16 (2H, m, CH₂), 3.95 (3H, s, N—CH₃), 3.90 (6H, s, N—CH₃, O—CH₃), 3.84 (3H, s, O—CH₃), 3.67-3.71 (1H, m, H-11a), 3.54-3.57 (2H, m, Hr₂-3), 2.53-2.56 (2H, m, CH₂), 2.23-2.30 (4H, m, CH₂), 2.00-2.05 (2H, m); ¹³C-NMR (125 MHz, CDCl₃): δ 169.8, 164.5, 162.6, 161.6, 159.5, 150.7, 147.9, 140.8, 133.1, 132.2, 132.1, 131.9, 131.7, 128.5, 128.4, 125.9, 125.5, 123.7, 123.1, 121.5, 120.7, 120.4, 119.7, 114.7, 112.0, 111.4, 103.9, 68.1, 56.2, 53.7, 51.0, 46.7, 36.8, 33.2, 29.6, 25.1, 24.1; HRMS m/z (+EI) calc. for C₃₆H₃₈N₆O₇ (M)⁺ 667.2880. found 667.2882 ([M+H]⁺, δ 0.3 ppm).

Compounds 14b, 15f and 16f, were made in an analogous manner, reacting compound 13 with the imidazolyl building block, followed by reaction with the MPB building block, and finally by deprotection.

(b) (S)-methyl 4-(4-(4-(4-(7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (16f)

[α]^(22.7) _(D)+188 (c=0.052, CHCl₃), IR (FTIR, v_(max)/cm⁻¹): 3301, 2169, 2136, 2018, 1978, 1937, 1680, 1564, 1518, 1439, 1265, 1181, 1108, 750, 722; ¹H-NMR (500 MHz, CDCl₃): δ 8.90 (1H, s, NH), 7.98 (1H, s, NH), 7.67 (1H, s, H-6), 7.63 (1H, d, J=4.4 Hz, H-11), 7.59 (2H, d, J=8.4 Hz, 2Ar—H), 7.46 (2H, d, J=8.4 Hz, 2Ar—H), 7.42 (1H, s, Im-H), 7.19 (1H, d, J=2.0 Hz, Py-H), 7.06 (1H, d, J=1.6 Hz, Py-H), 6.83 (1H, s, H-9), 4.10-4.22 (2H, m, CH₂), 4.07 (3H, s, N—CH₃), 3.96 (6H, s, N—CH₃, O—CH₃), 3.84 (3H, s, O—CH₃), 3.67-3.70 (1H, m, H-11a), 3.54-3.58 (2H, m, H₂-3), 2.57-2.67 (2H, m, CH₂), 2.26-2.31 (4H, m, CH₂), 1.98-2.05 (2H, m); ¹³C-NMR (125 MHz, CDCl₃): δ 169.5, 164.5, 162.5, 161.6, 156.4, 150.4, 147.8, 135.6, 132.1, 131.9 (2C), 128.5, 128.4, 126.0, 125.6, 123.5, 123.1, 121.5, 119.7, 114.6 (2C), 111.6, 111.0, 67.7, 56.1, 53.7, 51.1, 46.6, 36.9, 35.8, 33.9, 29.6, 24.7, 24.1; HRMS m/z (+EI) calc. for C₃₅H₃₇N₇O₇ (M)⁺ 668.2833. found 668.2838 [M+H]⁺, δ 0.5 ppm.

Example 3

The cytotoxicity potential of the compounds of the invention 16c-h was compared with the comparative compounds 16a, 16b and GWL-78 in a number of tumour cell lines and the non-cancer cell line WI38 after 96 hours exposure using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay, as described below.

A panel of several types of human cancer cell lines including epidermoid (A431), lung (A549), ovarian (A2780) and breast (MCF7 and MDAMB-231), as well as the non-tumour cell line WI38, were used to determine the cytotoxicity of the compounds. The cells were grown in normal conditions at 37° C. under a 5% CO₂ humidified atmosphere, either in Dulbecco's Modified Eagle Medium or Modified Eagle Medium (depending on the cell line), supplemented with 10% fetal bovine serum (Biosera, UK), 1% L-glutamine, 1% non-essential amino acids and 0.05% hydrocortisone (Gibco, Invitrogen, USA). Cells were then seeded into 96-well plates in a total volume of 160 μl, and allowed to reach a 30-40% degree of confluence before starting the experiment. The ligands were dissolved in sterilized ultrapure water at a maximum concentration of 100 μM, and serial decimal dilutions prepared. These were added to the cells in a volume of 40 μl. After 96 hours of continuous exposure to each ligand, the cytotoxicity was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Lancaster Synthesis Ltd, UK) colorimetric assay (Skehan P, S. R., et al., Journal of the National Cancer Institute 1990, 82, 1107). Absorbance was quantified by spectrophotometry at A=570 nm (ELx808, Bio-Tek Instruments, Inc., USA). IC₅₀ values were calculated by a dose-response analysis using the Origin 6.0™ software.

IC₅₀ (nM) Compound A431 A549 A2780 MCF7 MDAMB-231 Mia Paca 2 WI38 16a* 2.31 7.50 1.87 1.91 2.11 1.2 159.9 16b* 2.91 0.54 0.56 0.90 0.46 0.35 158.7 16c 4.60 0.019 0.96 1.70 2.70 0.34 425.9 I6d 0.19 0.47 0.15 0.37 0.45 0.11 1240 16e 0.0056 0.056 0.013 0.00002 0.000065 0.0013 473.8 16f 0.018 0.034 0.021 0.00002 0.00018 0.0021 129.2 16g 0.86 2.3 0.24 0.31 0.59 0.31 41.3 16h 03064 0.45 ND 0.075 0.015 0.25 65.6 GWL-78* 0.55 6.1 0.57 0.32 0.12 2.4 41.3 *comparative compounds

The cytotoxicity potential of the compounds of the invention 16c-h was also compared with the comparative compounds 16a, 16b and GWL-78 in a primary CLL cell lines using the Annexin V assay (van Engeland, M, et al., Cytometry 1998, 31, 1-9), as described below.

Freshly isolated peripheral blood CLL cells (1×10⁸ mL⁻¹) were cultured in RPMI medium (Invitrogen, Paisley, U.K.) supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal calf serum. The cells were incubated at 37° C. in a humidified 5% CO₂ atmosphere in the presence of each compound. All compounds were dissolved in DMSO and were evaluated in serial dilutions against the primary CLL cells. In addition, control cultures were carried out to which no drug was added. The cytotoxic effects of the compounds were quantified using an annexin V/propidium iodide flow cytometry assay (Bender Medsystems, Vienna, Austria). All assays were performed in duplicate, and LD₅₀ values were calculated from sigmoidal dose-response curves using the Prism 6.0 software (Graphpad Software Inc., San Diego, Calif.). The sigmoidal dose-response curves were derived by plotting log [compound concentration] against the percentage apoptosis induced by that concentration. A wide range of concentrations were used to establish the biologically active range for each individual compound.

Compound IC₅₀ (nM) 16a* 6.2 16b* 4.7 16c 2.1 I6d 3.0 16e 0.098 16f 0.17 16g 0.96 16h 0.037 GWL-78* 1.3

Example 4

16e and 16f were tested in in vivo xenograft studies in mouse models of both breast and pancreatic cancer.

Initially a small scale study was carried out to determine the MTD (maximum tolerated dose) in Swiss-Webster mice using intraperitoneal dosing (IP). The compounds were generally well tolerated without any signs of toxicity. However, a minor weight loss was observed at a dose level of 400 μg/Kg/day dose level for 16e (FIG. 1A). Some insignificant weight loss for 16f was also observed at the same dose level. A repeat MTD experiment using only 16f at 350 μg/Kg/day did not show any weight loss or other signs of toxicity (FIG. 1B). As 16f provided a marginally better toxicity profile in the MTD study, it was decided to carry out more extensive in vivo tumour xenograft studies on this molecule.

ER-Negative MDA MB 231 Breast Cancer Xenograft Study

In vivo studies of the activity of 16f were carried out in an ER-negative MDA MB 231 breast cancer xenograft in a mouse model. The human breast cancer cell line MDA-MB-231 (5×10⁸ cells) was employed to establish xenografts in the flanks of female MF1 nude mice, 2-3 months old and weighing 20-25 g. Subsequent passaging was by the subcutaneous implantation of small tumour pieces (approx 1 mm³) into the flank. When the tumours reached approx. 0.06 cm³ (three weeks post implantation) they were divided into 3 groups of 4 mice. The drug treated groups were administered with an IV dose (in DMSO) of either 250 μg/Kg/day or 300 μg/Kg/day for 5 consecutive days followed by two drug free days for 3 weeks, followed by 2 consecutive days in week 4 at which point the dosage was stopped. As shown in FIG. 2, 16f produced prominent in vivo antitumour activity compared to control mice (▴) at both 250 μg/Kg (♦) and 300 μg/Kg (▪) dose levels without any signs of toxicity (FIG. 2, where the arrow shows the last injected dose). The tumour did not re-grow up to 3 weeks after administration of the last IV dose in the case of the 300 μg/Kg dose level.

Mia Paca 2 Pancreatic Cancer Xenograft in Mouse Model

An in vivo study of 16f was carried out in a pancreatic cancer xenograft mouse model, in a similar manner to the above. The drug (16f) treated group was administered an IV dose of 300 μg/Kg/day for 5 consecutive days followed by 2 drug free days, and the cycle was continued for 3 weeks. 16f produced prominent antitumour activity compared to control mice at the 300 μg/Kg dose level without any signs of toxicity (FIG. 3: ♦300 μg/Kg 16f; ▪ control). The inability of the tumour to grow back immediately after withdrawing drug was notable, and no growth was observed up to 21 days after the last administered dose. Cross-sections from the tumour and control tissues were subjected to immunohistochemical staining, and the findings were consistent with NFκB inhibition in the experimental animals compared to controls.

Example 5

16f was evaluated in a commercial Transcription Factor Activation Profiling Array Assay™ (Signosis) using the HeLa cell line. In this assay the activities of 48 transcription factors could be monitored simultaneously using a collection of biotin-labeled DNA probes based on the consensus sequences of individual transcription factor DNA-binding sites. The top five transcription factors whose activities were at least 30% down-regulated by 7 h at a concentration of 10 nM for 4 hours were: NFAT, EGR, NF-κB, SMAD and OCT-4. The activity of NF-κB was reduced by almost 50%.

Based on the hypothesis that 16f may down-regulate the expression of NF-κB-dependent genes (e.g., IκB, BCL2, BCLX_(L)) by binding to the cognate DNA sequence of NF-κB, thereby blocking interaction of the transcription factor protein and inhibiting transcription of a number of genes, it was decided to explore this possibility in CLL cells in which NF-κB signaling is known to be active and closely correlates with the initiation and progression of malignancy. Using levels of phosphorylated IκB and p65 as surrogates for NF-κB activity in comparison to the Actin protein as a control, Western Blotting indicated that, after 24 hours incubation, 16f caused a significant suppression of phosphorylated IκBα at concentrations down to 0.1 nM, with only a marginal effect on phosphorylated-p65. 

The invention claimed is:
 1. A compound of formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein: the dotted double bond indicates the presence of a single or double bond between C2 and C3; R² is selected from —H, —OH, ═O, ═CH₂, —CN, —R, OR, halo, dihalo, ═CHR, ═CRR′, —O—SO₂—R, CO₂R and COR; R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo; where R and R′ are independently selected from optionally substituted C₁₋₇ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups; R¹⁰ and R¹¹ either together form a double bond, or are selected from H and QR^(Q) respectively, where Q is selected from O, S and NH and R^(Q) is H or C₁₋₇ alkyl or H and SO_(x)M, where x is 2 or 3, and M is a monovalent pharmaceutically acceptable cation; A is either:

where X and Y are selected from: CH and NMe; C—OH and NMe; CH and S; N and NMe; N and S; B is either a single bond or:

Where X and Y are as defined above; and R¹ is C₁₋₄ alkyl.
 2. A compound according to claim 1, where in group A, X and Y are selected from CH and NMe; and N and NMe.
 3. A compound according to claim 1, wherein B is a single bond.
 4. A compound according to claim 1, where B is B1 and in group B1, X and Y are selected from CH and NMe; and N and NMe.
 5. A compound according to claim 1, wherein R⁷ is selected from H and OR.
 6. A compound according to claim 5, wherein R⁷ is OR^(7A), where R^(7A) is optionally substituted C₁₋₇ alkyl.
 7. A compound according to claim 6, wherein R^(7A) is selected from Me, CH₂Ph and allyl.
 8. A compound according to claim 1, wherein R¹⁰ and R¹¹ form a double bond together.
 9. A compound according to claim 1, wherein R¹ is methyl.
 10. A compound according to claim 1, wherein R² is selected from —H, ═CH₂, —R, ═CHR, and ═CRR′.
 11. A compound according to claim 1, wherein R² is of the configuration C1:


12. A compound according to claim 1, wherein R² is selected from optionally substituted phenyl, optionally substituted naphthyl, optionally substituted pyridyl, optionally substituted quinolinyl, optionally substituted thienyl, optionally substituted furanyl, or isoquinolinyl.
 13. A compound according to claim 12, wherein R² group bears one to three substituent groups.
 14. A compound according to claim 12, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thienyl.
 15. A compound according to claim 12, wherein R² is selected from 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthienyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.
 16. A compound according to claim 1, wherein R² is selected from: (a) C₁₋₅ saturated aliphatic alkyl; (b) C₃₋₆ saturated cycloalkyl; (c)

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where the total number of carbon atoms in the R² group is no more than 5; (d)

wherein one of R^(25a) and R^(25b) is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and (e)

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃ alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
 17. A compound according to claim 1, wherein if R² is selected from ═O, ═CH₂, ═CHR, ═CRR′, there is a single bond between C2 and C3.
 18. A compound according to claim 1, wherein there is no double bond between C2 and C3 and R² is H.
 19. A pharmaceutical composition comprising a compound according to claim 1, and a pharmaceutically acceptable carrier or diluent.
 20. A method of treatment of a patient suffering from a proliferative disease, comprising administering to said patient a therapeutically acceptable amount of a compound according to claim 1, wherein the proliferative disease is selected from epidermoid cancer, lung cancer, ovarian cancer, breast cancer, chronic lymphocytic leukemia, and pancreatic cancer. 